CN114807077A - NphB mutant and application thereof - Google Patents

NphB mutant and application thereof Download PDF

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CN114807077A
CN114807077A CN202110134613.4A CN202110134613A CN114807077A CN 114807077 A CN114807077 A CN 114807077A CN 202110134613 A CN202110134613 A CN 202110134613A CN 114807077 A CN114807077 A CN 114807077A
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王舒
田振华
吴燕
包文艳
邢丽彤
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Ecolab Biotechnology Shanghai Co ltd
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    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
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Abstract

The invention discloses a mutant of NphB aromatic prenyltransferase and application thereof, wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 9 or SEQ ID NO. 11. The invention also discloses a preparation method of the cannabigerolic acid, which comprises the following steps: in a reaction system, the mutant is utilized to catalyze geranyl pyrophosphate and olivinic acid to generate cannabigerol acid. The invention improves the enzyme activity of the aromatic prenyltransferase and simultaneously improves the yield of CBGA.

Description

NphB mutant and application thereof
Technical Field
The invention relates to the technical field of biology, and in particular relates to a NphB mutant, a preparation method of cannabigerolic acid and application of the NphB mutant in preparation of cannabigerolic acid.
Background
Cannabinoids are a class of compounds with a variety of biological activities, which can be divided into three classes: (1) endocannabinoids, which occur naturally in the human body and can be produced in the human body; (2) phytocannabinoids, present only in the cannabis plant; (3) cannabinoids were synthesized in the laboratory. Cannabinoids have been shown to have several beneficial medical/therapeutic effects and thus cannabinoids are an active area of research in the pharmaceutical industry and may be used as pharmaceutical products for a variety of different diseases.
More than 80 phytocannabinoids are currently known, and include mainly tetrahydrocannabinol (THC, hallucinogenic addictive ingredient), cannabidiol (CBD, non-psychotropic active ingredient), cannabichromene (CBC), Cannabinol (CBN), Cannabigerol (CBG), and the like.
Cannabinoids are formed by decarboxylation of acidic cannabinoids under heat or long term storage conditions, such as tetrahydrocannabinolic acid (THCA) to form THC, cannabidiolic acid (CBDA) to form CBD and cannabichromenic acid (CBCA) to form CBC. While CBGA is a branched-point intermediate that is converted to an acid cannabinoid such as THCA, CBDA or CBCA by an oxidative cyclase, the reaction pathway is as follows, and thus CBGA is critical for the production of THC, CBD, CBC.
Figure BDA0002922965810000021
CBGA is mostly synthesized in the prior art by an enzymatic method catalyzed by Aromatic prenyltransferase (PTase) with olive acid and geranyl diphosphate (Gpp), such as the Aromatic prenyltransferase from Cannabis, which is named CsPT1 (canabis sativa prenyltransferase 1), as disclosed in US20150128301a1, by the university of sambuch. In addition, LIBREDE also inserted CsPT1 or a similar gene thereof into yeast (s. cerevisiae) in US20200017889a1, thereby converting olivic acid into CBGA.
Chinese patent CN110892075A also discloses a method for producing cannabinoids in genetically modified yeast containing NphB (hydroxyynaphhalene PTAse), a wild-type NphB, from the Streptomyces species CL190 strain (Bonitz et al, PLos ONE, 2011, 6(11), e 27336; Kuzuyama et al, 2005, Nature, 435, 983-. NphB (Q161A) produced more CBGA than the wild-type NphB (mundendam 2015). DE102018117233A1 discloses a method for the production of CBGA by introducing a gene encoding a modified NphB mutant into a host organism and expressing it. Wherein the synthesis amount of CBGA of modified NphB mutants (Q295F and Q295H) is remarkably increased compared with that of the wild-type NphB (SEQ ID NO:2 in the patent document), and CBGA is increased by 20 times compared with 2-O-geranyl olivinic acid.
However, the activity of the prior art aromatic prenyl transferase enzymes still remains to be improved to produce CBGA in as large an amount as possible.
Disclosure of Invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide an improved aromatic prenyl transferase, in particular an improved NphB enzyme, for improved CBGA production.
Figure BDA0002922965810000031
In a first aspect of the invention there is provided a mutant of an NphB aromatic prenyltransferase, the amino acid sequence of which is shown in SEQ ID No. 9 or SEQ ID No. 11.
Preferably, the nucleotide sequence encoding the mutant is shown as SEQ ID NO. 10 or SEQ ID NO. 12.
In a second aspect, the present invention provides an isolated nucleic acid encoding a mutant according to the first aspect of the invention.
In a third aspect, the invention provides a recombinant expression vector comprising the isolated nucleic acid of the second aspect of the invention; the recombinant expression vector is preferably the pET28a plasmid.
In a fourth aspect, the invention provides a transformant comprising an isolated nucleic acid according to the second aspect of the invention, or a recombinant expression vector according to the third aspect of the invention; the host used for the construction of the transformant is preferably Escherichia coli, for example E.coli BL21(DE 3).
In a fifth aspect, the present invention provides a method for preparing the mutant of the first aspect, which comprises culturing the transformant of the fourth aspect to obtain a fermentation product, and obtaining the mutant from the fermentation product;
the method preferably further comprises the step of purifying the mutant;
the method more preferably uses a Ni column to purify the mutant.
The sixth aspect of the present invention provides a method for preparing cannabigerolic acid, which comprises the following steps: in a reaction system, the mutant of the first aspect of the invention is used for catalyzing geranyl pyrophosphate and olive alcohol acid to generate cannabigerolic acid.
Preferably, the reaction system comprises 1 mM-10 mM geranyl pyrophosphate, preferably 2.5mM or 5 mM; 2mM to 20mM, preferably 5mM or 10mM, of olivinic acid.
In a preferred embodiment, the mutant has a final concentration of 0.5g/mL to 1.5g/mL, preferably 1 g/mL;
the reaction system comprises MgCl with the concentration of 1 mM-10 mM 2 Preferably 5mM MgCl 2 ；
The pH value of the reaction system is 7.5-8.5, and 8.0 is preferred;
the catalysis is carried out at 30-40 ℃, and preferably at 30 ℃;
the catalysis time is 1-6 hours.
In a sixth aspect, the invention provides the use of a mutant according to the first aspect of the invention, an isolated nucleic acid according to the second aspect of the invention, a recombinant expression vector according to the third aspect of the invention or a transformant according to the fourth aspect of the invention for the preparation of cannabigerolic acid or cannabinoid.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the enzyme activity of the aromatic prenyltransferase is improved, and the yield of CBGA is improved.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The experimental methods in the invention are conventional methods unless otherwise specified, and the gene cloning operation can be specifically referred to the molecular cloning experimental guidance compiled by J. Sambruka et al.
The abbreviations for the amino acids in the present invention are those conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 1.
TABLE 1
Figure BDA0002922965810000051
Codons corresponding to the amino acids are also conventional in the art, and the correspondence between specific amino acids and codons is shown in table 2.
TABLE 2
Figure BDA0002922965810000052
Figure BDA0002922965810000061
Plasmid pBAD was purchased from Novagen; DpnI, NdeI, HindIII enzymes were purchased from Thermo Fisher; the Exnase II enzyme was purchased from Nanjing Novozam Biotech Ltd; coli BL21(DE3) competent cells were purchased from changsheng biotechnology, llc, beijing dingguo; olivinic Acid (OA) was synthesized by the laboratory; geranyl pyrophosphate (GPP) from sigma; the plasmid extraction kit was purchased from Biotechnology engineering (Shanghai) GmbH.
HPLC detection method: and (3) chromatographic column: agilent Eclipse plus C18(3.5 μm, 150X 4.6 mm); mobile phase: the mobile phase A is 0.1% TF water solution, and the mobile phase B is 0.1% TFA acetonitrile solution; gradient elution: 90% A + 10% B (0min), 100% B (10min), 100% B (11min), 90% A + 10% B (11.5min), 90% A + 10% B (16 min); detection wavelength: 210nm/254 nm; flow rate: 1 mL/min; sample introduction volume: 10 mu L of the solution; column temperature: at 30 ℃.
EXAMPLE 1 construction of NphB mutants
The primer sequences designed by constructing the mutant library of the wild type NphB of the sequence table at the 49 th, 286 th and 288 th sites of SEQ ID NO. 1 through point mutation are shown in the table 3, and the combined mutation can be obtained through the combined amplification of the primers in the table.
TABLE 3
Figure BDA0002922965810000062
Figure BDA0002922965810000071
The NphB gene is synthesized according to the sequence of SEQ ID NO. 1 in the sequence table, the gene synthesis company is completed by Suzhou Jinweizhi Biotechnology GmbH (Suzhou industrial park, Star lake street 218, biological nanometer technology park, floor C3), and the PDB number of the NphB coded by the SEQ ID NO. 1 is 1ZB 6. Then, NdeI and HindIII restriction sites were introduced into plasmid pET28a to construct plasmid pET28 a-NphB. The plasmid pET28a-NphB was used as a template to amplify the desired band by PCR.
The PCR amplification system is as follows:
reagent Dosage (mu L)
2 × PCR buffer (with high fidelity enzyme) 25
Primer F 1
Primer R 1
Form panel 1
Deionized water 22
The PCR amplification procedure was as follows:
Figure BDA0002922965810000072
Figure BDA0002922965810000081
the PCR product was digested with DpnI at 37 ℃ for 2 hr. After completion of the reaction, the cells were transformed into E.coli BL21(DE3) competent cells, plated on LB medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ and harvested to obtain transformants containing various mutants.
Example 2 expression of NphB mutants
After the engineering bacteria containing mutant genes are activated by plating and streaking, a single colony is selected and inoculated into 5ml LB liquid culture medium containing 50 mug/ml kanamycin, and shake culture is carried out for 12h at 37 ℃. Transferred to 50ml of fresh LB liquid medium containing 50. mu.g/ml kanamycin at an inoculum size of 2 v/v%, shake-cultured at 37 ℃ until OD600 reaches about 0.8, and IPTG was added to a final concentration of 0.1mM, and induced-cultured at 25 ℃ for 16 hours. After the culture was completed, the culture was centrifuged at 10000rpm for 10min, and the supernatant was discarded to collect the cells.
Example 3 purification of NphB mutants
The bacterial sludge of each mutant was washed twice with 0.1M phosphate buffer (containing 150mM NaCl) at pH 8.0, and then homogenized and disrupted at a low temperature and high pressure in a ratio of 1:5, and the disrupted solution was centrifuged to remove the precipitate, and the resulting supernatant was a crude enzyme solution containing each recombinant enzyme. The crude enzyme solution was then purified using a Ni column. Protein concentration was determined by the Bradford method and dispensed into 2mL centrifuge tubes and stored in a-80 ℃ freezer.
Example 4 Activity assay of NphB mutants
The enzyme activity was measured by the following method. The reaction system was charged with geranyl pyrophosphate (GPP), Olivinic Acid (OA), and MgCl at a final concentration of 2.5mM, 5mM 2 50mM Tris pH 8.0, and finally adding the NphB mutant enzyme solution with the final concentration of 1mg/mL to start the reaction, wherein the final volume of the reaction is 100 mu L. After 1 hour of reaction at 30 ℃, 40. mu.L of the reaction mixture was quenched by adding 80. mu.L of acetonitrile. The samples were centrifuged at 12000rpm for 5min to remove protein precipitate, and the supernatant was subjected to HPLC for determination of substrate and product concentrations, and enzyme activity was calculated. As shown in Table 4, as a control, the sequence corresponding to Enz.16 is SEQ ID NO:12 in patent CN110892075A, and the sequences corresponding to Enz.17 and Enz.18 are mutants in patent DE102018117233A 1.
Definition of unit enzyme activity: the amount of enzyme required to produce 1. mu. moL of CBGA per minute under the specified reaction conditions (30 ℃).
The enzyme activity is improved by multiple: the ratio of the mutant enzyme activity to the wild type enzyme activity.
TABLE 4
Figure BDA0002922965810000091
The sequence at "/" can be deduced from SEQ ID NO 1-20.
Example 5 enzymatic Synthesis of CBGA
The enzyme-catalyzed system was carried out as follows. The final concentration of geranyl pyrophosphate (GPP), 10mM Olivinic Acid (OA), and 5mM MgCl was added to the reaction system 2 50mM Tris pH 8.0, and finally 1mg/mL Enz.01, Enz.04, and Enz.05 were added to the reaction mixture to start the reaction, the final volume of the reaction was 1 mL. After 6 hours of reaction at 30 ℃, 40. mu.L of the reaction mixture was quenched by adding 80. mu.L of acetonitrile. Samples were centrifuged at 12000rpm for 5min to remove protein precipitate and supernatants were assayed for substrate and product concentrations by HPLC. The results show that the CBGA concentration in the Enz.01, Enz.04 and Enz.05 reaction systems respectively reaches 1.10g/L, 1.42g/L and 1.51 g/L.
SEQUENCE LISTING
<110> Chongkola Biotechnology (Shanghai) Ltd
<120> NphB mutant and application thereof
<130> P20017665C
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 307
<212> PRT
<213> Artificial Sequence
<220>
<223> wild type NphB sequence
<400> 1
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Tyr
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 2
<211> 924
<212> DNA
<213> Artificial Sequence
<220>
<223> wild type NphB sequence
<400> 2
atgtctgaag ctgctgatgt tgaaagagtt tacgctgcta tggaagaagc tgctggtttg 60
ttgggtgttg cttgtgctag agataagatc tacccattgt tgtctacttt tcaagatact 120
ttggttgaag gtggttctgt tgttgttttt agtatggctt ctggtagaca ttctactgaa 180
ttggattttt ctatttctgt tccaacttct catggtgacc catacgctac tgttgttgaa 240
aagggtttgt ttccagctac tggtcatcca gttgatgatt tgttggctga tactcaaaag 300
catttgccag tttctatgtt tgctatcgat ggtgaagtta caggtggttt taagaaaact 360
tacgctttct tcccaactga taacatgcca ggtgttgctg aattgtctgc tattccatct 420
atgccaccag ctgtcgctga aaacgctgaa ttgtttgcta gatacggttt ggataaggtt 480
caaatgactt ctatggatta caaaaagaga caagtcaact tgtacttttc tgaattgtct 540
gctcaaactt tggaagctga atctgtcttg gctttggtta gagaattggg tttgcatgtt 600
ccaaacgaat tgggtttgaa gttttgtaag agatcctttt cagtgtaccc tactttgaac 660
tgggaaactg gtaaaattga tagattgtgt tttgctgtta tttctaatga tccaactttg 720
gttccatctt ctgatgaagg tgacattgaa aagtttcata actacgctac taaggctcca 780
tacgcttacg ttggtgaaaa gagaactttg gtttacggtt tgactttgtc tccaaaggaa 840
gaatactaca agttgggtgc ttactaccat attactgatg ttcaaagagg tttgttgaag 900
gcttttgatt ctttggaaga ttaa 924
<210> 3
<211> 307
<212> PRT
<213> Artificial Sequence
<220>
<223> G286S/Y288A sequence
<400> 3
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Ala
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 4
<211> 924
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S/Y288A sequence
<400> 4
atgtctgaag ctgctgatgt tgaaagagtt tacgctgcta tggaagaagc tgctggtttg 60
ttgggtgttg cttgtgctag agataagatc tacccattgt tgtctacttt tcaagatact 120
ttggttgaag gtggttctgt tgttgttttt agtatggctt ctggtagaca ttctactgaa 180
ttggattttt ctatttctgt tccaacttct catggtgacc catacgctac tgttgttgaa 240
aagggtttgt ttccagctac tggtcatcca gttgatgatt tgttggctga tactcaaaag 300
catttgccag tttctatgtt tgctatcgat ggtgaagtta caggtggttt taagaaaact 360
tacgctttct tcccaactga taacatgcca ggtgttgctg aattgtctgc tattccatct 420
atgccaccag ctgtcgctga aaacgctgaa ttgtttgcta gatacggttt ggataaggtt 480
caaatgactt ctatggatta caaaaagaga caagtcaact tgtacttttc tgaattgtct 540
gctcaaactt tggaagctga atctgtcttg gctttggtta gagaattggg tttgcatgtt 600
ccaaacgaat tgggtttgaa gttttgtaag agatcctttt cagtgtaccc tactttgaac 660
tgggaaactg gtaaaattga tagattgtgt tttgctgtta tttctaatga tccaactttg 720
gttccatctt ctgatgaagg tgacattgaa aagtttcata actacgctac taaggctcca 780
tacgcttacg ttggtgaaaa gagaactttg gtttacggtt tgactttgtc tccaaaggaa 840
gaatactaca agttgtctgc tgcttaccat attactgatg ttcaaagagg tttgttgaag 900
gcttttgatt ctttggaaga ttaa 924
<210> 5
<211> 307
<212> PRT
<213> Artificial Sequence
<220>
<223> V49S/G286S/Y288A sequence
<400> 5
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Ser Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Ala
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 6
<211> 924
<212> DNA
<213> Artificial Sequence
<220>
<223> V49S/G286S/Y288A sequence
<400> 6
atgtctgaag ctgctgatgt tgaaagagtt tacgctgcta tggaagaagc tgctggtttg 60
ttgggtgttg cttgtgctag agataagatc tacccattgt tgtctacttt tcaagatact 120
ttggttgaag gtggttctgt tgtttctttt agtatggctt ctggtagaca ttctactgaa 180
ttggattttt ctatttctgt tccaacttct catggtgacc catacgctac tgttgttgaa 240
aagggtttgt ttccagctac tggtcatcca gttgatgatt tgttggctga tactcaaaag 300
catttgccag tttctatgtt tgctatcgat ggtgaagtta caggtggttt taagaaaact 360
tacgctttct tcccaactga taacatgcca ggtgttgctg aattgtctgc tattccatct 420
atgccaccag ctgtcgctga aaacgctgaa ttgtttgcta gatacggttt ggataaggtt 480
caaatgactt ctatggatta caaaaagaga caagtcaact tgtacttttc tgaattgtct 540
gctcaaactt tggaagctga atctgtcttg gctttggtta gagaattggg tttgcatgtt 600
ccaaacgaat tgggtttgaa gttttgtaag agatcctttt cagtgtaccc tactttgaac 660
tgggaaactg gtaaaattga tagattgtgt tttgctgtta tttctaatga tccaactttg 720
gttccatctt ctgatgaagg tgacattgaa aagtttcata actacgctac taaggctcca 780
tacgcttacg ttggtgaaaa gagaactttg gtttacggtt tgactttgtc tccaaaggaa 840
gaatactaca agttgtctgc tgcttaccat attactgatg ttcaaagagg tttgttgaag 900
gcttttgatt ctttggaaga ttaa 924
<210> 7
<211> 307
<212> PRT
<213> Artificial Sequence
<220>
<223> V49S/G286S/Y288N sequence
<400> 7
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Ser Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Asn
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 8
<211> 924
<212> DNA
<213> Artificial Sequence
<220>
<223> V49S/G286S/Y288N sequence
<400> 8
atgtctgaag ctgctgatgt tgaaagagtt tacgctgcta tggaagaagc tgctggtttg 60
ttgggtgttg cttgtgctag agataagatc tacccattgt tgtctacttt tcaagatact 120
ttggttgaag gtggttctgt tgtttctttt agtatggctt ctggtagaca ttctactgaa 180
ttggattttt ctatttctgt tccaacttct catggtgacc catacgctac tgttgttgaa 240
aagggtttgt ttccagctac tggtcatcca gttgatgatt tgttggctga tactcaaaag 300
catttgccag tttctatgtt tgctatcgat ggtgaagtta caggtggttt taagaaaact 360
tacgctttct tcccaactga taacatgcca ggtgttgctg aattgtctgc tattccatct 420
atgccaccag ctgtcgctga aaacgctgaa ttgtttgcta gatacggttt ggataaggtt 480
caaatgactt ctatggatta caaaaagaga caagtcaact tgtacttttc tgaattgtct 540
gctcaaactt tggaagctga atctgtcttg gctttggtta gagaattggg tttgcatgtt 600
ccaaacgaat tgggtttgaa gttttgtaag agatcctttt cagtgtaccc tactttgaac 660
tgggaaactg gtaaaattga tagattgtgt tttgctgtta tttctaatga tccaactttg 720
gttccatctt ctgatgaagg tgacattgaa aagtttcata actacgctac taaggctcca 780
tacgcttacg ttggtgaaaa gagaactttg gtttacggtt tgactttgtc tccaaaggaa 840
gaatactaca agttgtctgc taattaccat attactgatg ttcaaagagg tttgttgaag 900
gcttttgatt ctttggaaga ttaa 924
<210> 9
<211> 307
<212> PRT
<213> Artificial Sequence
<220>
<223> V49G/G286S/Y288A sequence
<400> 9
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Gly Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Ala
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 10
<211> 924
<212> DNA
<213> Artificial Sequence
<220>
<223> V49G/G286S/Y288A sequence
<400> 10
atgtctgaag ctgctgatgt tgaaagagtt tacgctgcta tggaagaagc tgctggtttg 60
ttgggtgttg cttgtgctag agataagatc tacccattgt tgtctacttt tcaagatact 120
ttggttgaag gtggttctgt tgttggtttt agtatggctt ctggtagaca ttctactgaa 180
ttggattttt ctatttctgt tccaacttct catggtgacc catacgctac tgttgttgaa 240
aagggtttgt ttccagctac tggtcatcca gttgatgatt tgttggctga tactcaaaag 300
catttgccag tttctatgtt tgctatcgat ggtgaagtta caggtggttt taagaaaact 360
tacgctttct tcccaactga taacatgcca ggtgttgctg aattgtctgc tattccatct 420
atgccaccag ctgtcgctga aaacgctgaa ttgtttgcta gatacggttt ggataaggtt 480
caaatgactt ctatggatta caaaaagaga caagtcaact tgtacttttc tgaattgtct 540
gctcaaactt tggaagctga atctgtcttg gctttggtta gagaattggg tttgcatgtt 600
ccaaacgaat tgggtttgaa gttttgtaag agatcctttt cagtgtaccc tactttgaac 660
tgggaaactg gtaaaattga tagattgtgt tttgctgtta tttctaatga tccaactttg 720
gttccatctt ctgatgaagg tgacattgaa aagtttcata actacgctac taaggctcca 780
tacgcttacg ttggtgaaaa gagaactttg gtttacggtt tgactttgtc tccaaaggaa 840
gaatactaca agttgtctgc tgcttaccat attactgatg ttcaaagagg tttgttgaag 900
gcttttgatt ctttggaaga ttaa 924
<210> 11
<211> 307
<212> PRT
<213> Artificial Sequence
<220>
<223> V49G/G286S/Y288N sequence
<400> 11
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Gly Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Ser Ala Asn
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 12
<211> 924
<212> DNA
<213> Artificial Sequence
<220>
<223> V49G/G286S/Y288N sequence
<400> 12
atgtctgaag ctgctgatgt tgaaagagtt tacgctgcta tggaagaagc tgctggtttg 60
ttgggtgttg cttgtgctag agataagatc tacccattgt tgtctacttt tcaagatact 120
ttggttgaag gtggttctgt tgttggtttt agtatggctt ctggtagaca ttctactgaa 180
ttggattttt ctatttctgt tccaacttct catggtgacc catacgctac tgttgttgaa 240
aagggtttgt ttccagctac tggtcatcca gttgatgatt tgttggctga tactcaaaag 300
catttgccag tttctatgtt tgctatcgat ggtgaagtta caggtggttt taagaaaact 360
tacgctttct tcccaactga taacatgcca ggtgttgctg aattgtctgc tattccatct 420
atgccaccag ctgtcgctga aaacgctgaa ttgtttgcta gatacggttt ggataaggtt 480
caaatgactt ctatggatta caaaaagaga caagtcaact tgtacttttc tgaattgtct 540
gctcaaactt tggaagctga atctgtcttg gctttggtta gagaattggg tttgcatgtt 600
ccaaacgaat tgggtttgaa gttttgtaag agatcctttt cagtgtaccc tactttgaac 660
tgggaaactg gtaaaattga tagattgtgt tttgctgtta tttctaatga tccaactttg 720
gttccatctt ctgatgaagg tgacattgaa aagtttcata actacgctac taaggctcca 780
tacgcttacg ttggtgaaaa gagaactttg gtttacggtt tgactttgtc tccaaaggaa 840
gaatactaca agttgtctgc taattaccat attactgatg ttcaaagagg tttgttgaag 900
gcttttgatt ctttggaaga ttaa 924
<210> 13
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> V49S-Forward primer
<400> 13
ggtggttctg ttgtttcttt tagtatggct tctg 34
<210> 14
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> V49S-reverse primer
<400> 14
cagaagccat actaaaagaa acaacagaac cacc 34
<210> 15
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> V49G-Forward primer
<400> 15
ggtggttctg ttgttggttt tagtatggct tctg 34
<210> 16
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> V49G-reverse primer
<400> 16
cagaagccat actaaaacca acaacagaac cac 33
<210> 17
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S-Forward primer
<400> 17
gaatactaca agttgtctgc ttactaccat attac 35
<210> 18
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S-reverse primer
<400> 18
gtaatatggt agtaagcaga caacttgtag tattc 35
<210> 19
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> G286A-Forward primer
<400> 19
gaatactaca agttggctgc ttactaccat attac 35
<210> 20
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> G286A-reverse primer
<400> 20
gtaatatggt agtaagcagc caacttgtag tattc 35
<210> 21
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S/Y288A-Forward primer
<400> 21
gaatactaca agttgtctgc tgcttaccat attactgatg 40
<210> 22
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S/Y288A-reverse primer
<400> 22
catcagtaat atggtaagca gcagacaact tgtagtattc 40
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S/Y288N-Forward primer
<400> 23
gaatactaca agttgtctgc taattaccat attactgatg 40
<210> 24
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> G286S/Y288N-reverse primer
<400> 24
catcagtaat atggtaatta gcagacaact tgtagtattc 40
<210> 25
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Q161A-Forward primer
<400> 25
ggtttggata aggttgctat gacttctatg gat 33
<210> 26
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Q161A-reverse primer
<400> 26
atccatagaa gtcatagcaa ccttatccaa acc 33
<210> 27
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Q295F-Forward primer
<400> 27
catattactg atgtttttag aggtttgttg aag 33
<210> 28
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Q295F-reverse primer
<400> 28
cttcaacaaa cctctaaaaa catcagtaat atg 33
<210> 29
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Q295H-Forward primer
<400> 29
catattactg atgttcatag aggtttgttg aag 33
<210> 30
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Q295H-reverse primer
<400> 30
cttcaacaaa cctctatgaa catcagtaat atg 33

Claims (10)

1. A mutant of NphB aromatic prenyltransferase, wherein the amino acid sequence of the mutant is shown in SEQ ID NO. 9 or SEQ ID NO. 11.
2. The mutant according to claim 1, wherein the nucleotide sequence encoding the mutant is as set forth in SEQ ID NO 10 or SEQ ID NO 12.
3. An isolated nucleic acid encoding the mutant of claim 1 or 2.
4. A recombinant expression vector comprising the isolated nucleic acid of claim 3; the recombinant expression vector is preferably the pET28a plasmid.
5. A transformant comprising the isolated nucleic acid of claim 3, or the recombinant expression vector of claim 4; the host used for constructing the transformant is preferably Escherichia coli, for example, E.coli BL21(DE 3).
6. A method for producing the mutant of claim 1 or 2, which comprises culturing the transformant of claim 5 to obtain a fermentation product, and obtaining the mutant from the fermentation product; preferably, further comprising the step of purifying the mutant; more preferably, the mutant is purified using a Ni column.
7. A method for preparing cannabigerolic acid, comprising the steps of: catalyzing geranyl pyrophosphate and olive alcohol acid to generate cannabigerolic acid by using the mutant as claimed in claim 1 or 2 in a reaction system.
8. The production method according to claim 7, wherein the reaction system comprises 1mM to 10mM of geranyl pyrophosphate, preferably 2.5mM or 5 mM;
and/or 2mM to 20mM, preferably 5mM or 10mM, of olive alcohol acid.
9. The production method according to claim 7 or 8, wherein the mutant has a final concentration of 0.5g/mL to 1.5g/mL, preferably 1g/mL, in the reaction system;
and/or, the reaction system comprises 1 mM-10 mM MgCl 2 Preferably 5mM MgCl 2 ；
And/or the pH value of the reaction system is 7.5-8.5, preferably 8.0;
and/or the catalysis is carried out at 30-40 ℃, preferably at 30 ℃;
and/or the catalysis time is 1-6 hours.
10. Use of the mutant of claim 1 or 2, the isolated nucleic acid of claim 3, the recombinant expression vector of claim 4, or the transformant of claim 5 for the preparation of cannabigerolic acid or cannabinoid.
CN202110134613.4A 2021-01-29 2021-01-29 NphB mutant and application thereof Pending CN114807077A (en)

Priority Applications (1)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110134613.4A CN114807077A (en) 2021-01-29 2021-01-29 NphB mutant and application thereof

Publications (1)

Publication Number Publication Date
CN114807077A true CN114807077A (en) 2022-07-29

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Family Applications (1)

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Country Status (1)

Country Link
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